The long-term objective of the proposed effort is to develop a new anti-HER3 therapeutic for concomitant administration with approved anti-HER2 therapies in HER2+ breast cancer (BCa) patients. Trastuzumab (TZMB), a monoclonal anti-HER2 antibody, and lapatinib, a dual HER2/EGFR tyrosine kinase inhibitor, are FDA approved to treat HER2+ BCa. Unfortunately, TZMB fails to block ligand-induced HER2/HER3 heterodimerization and most patients either do not respond or eventually develop resistance. Similarly, clinical utility of lapatinib monotherapy is limited due to treatment-induced feedback upregulation of HER3 expression and activity. Hence, HER3 critically modulates clinical efficacy of HER2-directed therapies, underscoring the need for effective anti-HER3 therapies. The overall goal of this Phase I feasibility project is to design, synthesize and evaluate the therapeutic efficacy of a bifunctional peptide that (i) blocks heterodimerization of HER3 with other ErbB family members (HER2, EGFR, HER4) and (ii) delivers a mitochondriotoxic Pro- Apoptotic-Peptide (PAP) to induce apoptosis in HER2+ BCa cells. Hence, this innovative therapeutic strategy simultaneously neutralizes HER3 activity and delivers PAP for added growth inhibition of targeted cells. We hypothesize this will lead to a more complete blockade of ErbB signaling and block/limit emergence of resistance to anti-HER2 therapy by inhibiting/limiting reactivation of HER3. We will test our hypothesis through the following two specific aims: (1) design a bifunctional anti-HER3 apoptotic peptide (AHAP) therapeutic that binds specifically to the heterodimerization domain of HER3; (2) evaluate the growth inhibitory effects of AHAP, with and without TZMB/lapatinib, in a panel of HER2+ BCa cell lines (TZMB sensitive/resistant). Growth inhibitory studies will be accompanied with co-immunoprecipitation, immunoblot and protein array (RTK and phospho-kinase) analyses to confirm that the bifunctional peptide inhibits HER2/HER3 dimerization and downstream signaling. These mechanistic studies will also allow us to test: (i) if there is synergism between anti-HER2 and anti-HER3 therapy in HER2+ TZMB sensitive breast cancer cells; (ii) if anti-HER3 therapy can restore, at least in part, responsiveness of resistant lines to TZMB and (iii) if the peptide can block reactivation of HER3 following lapatinib treatment and consequently, improve growth inhibition. Finally, we will evaluate the in vivo anti-tumor effects using a mouse xenograft model with a TZMB sensitive HER2+ BCa cell line. Successful completion of the proposed work will result in a novel anti-HER3 therapeutic that can effectively block HER2/HER3 heterodimerization. This strategy can potentially reduce treatment failures of anti-HER2 therapies and improve clinical outcome, thereby saving significant amounts in health care expenditure.